The transcription factor MRF4 is an important regulator of development, growth, and regeneration of skeletal muscle cells. Elucidating the mechanisms that control MRF4 gene transcription should increase our understanding of these processes. The frog Xenopus laevis is an attractive model system for comparing MRF4 expression patterns and transcriptional control mechanisms with those known in mammals because of the phylogenetic distance between them. One can also take advantage of the efficient procedures available for testing transcriptional regulation in transgenic X. laevis. In mammals, MRF4 expression is seen first in particular cells of the embryonic myotomes and later in myofibers in the limbs. Whether precisely the same pattern occurs in X. laevis myotomes and limbs has not yet been examined. In mouse, the regulatory elements responsible for MRF4 transcription in different embryonic and fetal myogenic cells are distributed over many kilobases of upstream sequence, but for the X. laevis MRF4a gene, only several hundred base pairs (bp) of the proximal promoter is needed for transgenic expression in muscle cells ranging from myotomes to body and limb myofibers in postmetamorphic frogs. Less than 200 bp within this region shows significant identity to any mammalian MRF4 gene promoter. This most-conserved region in XlMRF4a drove only low-level transgenic expression in myotomes and in tadpole trunk and tail muscle, but not in the limbs during metamorphosis. Based on these observations, the specific hypothesis of this application is that separable elements in the XlMRF4a promoter have distinct roles in different myotomal cell subpopulations and in limbs. The specific aims are to: 1) Determine, by in situ hybridization and sectioning, the precise cells that express endogenous MRF4, from the earliest myotome through postmetamorphic limb muscles. 2) Identify the specific regulatory sites in the XlMRF4a proximal promoter, with further deletions and point mutations, which are necessary and sufficient for transgenic expression by the myogenic cell populations identified in Aim 1. PUBLIC HEALTH RELEVANCE: The transcription factor MRF4 is an important regulator of development, growth, and regeneration of skeletal muscle cells. The frog Xenopus laevis is an attractive model for elucidating the mechanisms that control MRF4 gene transcription. The work proposed will determine which cells express MRF4, in the early embryo through postmetamorphic limb muscles, and identify the specific regulatory sites in the gene promoter that are necessary and sufficient for expression.